Agriculture is one of the main pillars of the economy of developing countries. In Brazil, the main crops produced are: soybeans, cotton, maize, beans, coffee beans, sugar-cane, among others. However, the production of most of these crops is impaired by the incidence of insect pests and diseases. The losses in the agricultural production, caused by insect pests reach high levels of over 37%, and it is known that about 200 plant diseases are caused by phytopathogens (HAQ, S. K., et al, Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. Archives of biochemistry and biophysics, v. 431 No. 1, p. 145-159; 2004). The need to control pest insects and pathogens ion agriculture has pushed the development of different pesticides, which decrease losses and contribute to the agribusiness. However, such pesticides are highly toxic, not only to the target species, but also to other animals and even to humans.
Cotton plant is, among all the domestic and cultivatable plants, one of the most widely attacked by diseases and pest insects, besides exhibiting high sensitivity to the occurrence imposed by weeds (Beltrão, E. M., Souza, J. G. O agronegócio do algodão no Brasil. Embrapa: Brasília, v. 01, 1999). Among the main pest insects, the “bicudo-do-algodoeiro” or boll weevil (Anthonomus grandis) stands out—(Boheman, C. H. Description of new species. In Schoenherr, Genera et species Curculionidum cum synonymia hujus Familiae, vol. 7, pt. 2. Paris: Roret. 461 p., 1843), which is considered one of the most serious pests in cotton cultivation and is widespread in Mexico, Cuba, Haiti, Venezuela, Colombia, Paraguay and Brazil. This insect uses the flower buds and fruits of its host as a source of food and development site, directly impairing the commercialization of cotton fiber. The infestation levels rise rapidly, and the damages may reach 100% of the production, if control measures are not adequate. This insect represents a great potential of damage, being considered a key-pest in the planning and control of insects that are harmful to the crop, chiefly due to the difficulty of control by chemical insecticides.
Cotton plant and its pests co-exist for a long period of evolution. Plant and insect form an interdependent and competitive morphologic and biochemical system, resulting, in most cases, ion the use of part of the plant by the insect. This part used represents the damage caused by the insect to the plant and depends on the population of the pest, as well as on the capability of the plant to resist the attack and to recover from the damage undergone (Beltrão, E. M., Souza, J. G. O agronegócio do algodão no Brasil. Embrapa: Brasília, v. 01, 1999).
The plant-insect interaction can be viewed in at least two ways: from the point of view of the insect, with the plant varying from adequate to completely inadequate as a host and, on the other hand, from the point of view of the plant, where the smaller the number of species and abundance of insects associated thereto, and the smaller the effect which these insects exert on them, the greater the resistance to these insects (Santos, W. J. Identificação, biologia, amostragem e controle das pragas do algodoeiro. In: Embrapa Agropecuária Oeste; Embrapa Algodão. Algodão: tecnologia de produção, p. 296-2002).
Between one extreme and the other of resistance of the plants to pest insects, there is a complex and extensive arsenal of mechanisms of attack and counterattack to the action of the insects, which goes from a simple morphologic impediment to complex phytochemical components that interfere directly with the metabolic process involved in using the plant as a host for the insect. In practical terms, the resistance of the cotton plant to pest insects represents the capability of certain cultivars to produce cotton of better quality in larger quantity that other cultivars under attack of the same population of pest insects (Freire, E. C. Cultivares e produção de semente na melhoria da qualidade do algodão no nordeste e centro-oeste do Brasil, Boletim informative Embrapa/CNPA, 1997).
At present, there is a need to develop more selective methods, without action on the environment, which are non-persistent, biodegradable and adapt well to insect pest control programs.
Due to the dangers associated to chemical control of insects, a number of molecular approaches for the control of pests on plants have been developed. Over the past thirty years the use of Bacillus thuringiensis has proved to be a safe alternative, and its endotoxins are more and more transferred to commercial varieties. Fields planted with transgenic varieties exhibit a drastic reduction in the use of insecticides all over the world, which additionally causes a decrease in the cases of poisoning and also an increase in the number of beneficial insects in the plantations.
At present, the interfering RNA is a tool with great potential for combating pest insects, in view of its specificity and high efficiency in suppressing the target mRNA.
The interference RNA mechanism (RNAi) is a phenomenon that occurs naturally in the cells and in various eukaryotic organisms. Such a process, described primarily on plants, was called post-transcriptional gene silencing, or PTGS (NAPOLI, C. et al, Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in Trans. Plant Cell, V. 2, 4. p. 279-289, 1990). However, the first description of genic silencing on animals, as well as the better understanding thereof, was obtained on Caenorhabditis elegans, free-life nematode and model organism (FIRE, A. et al, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, v. 391, n. 6669, p 806-811, 1998). At present, it is known that this process participates integrally in the regulation of the genic expression on various plants and other eukaryotes (LILLEY, C. J., et al, Recent progress in the development of RNA interference for plant parasitic nematodes. Molecular Plant Pathology, v. 8, n5. P. 701-711, 2007).
The action mechanism thereof is based chiefly on the introduction of a double-stranded RNA into a target organism, by micro injection or ingestion (FIRE, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, v. 391, n. 6669. p. 806-811. 1998). This double-stranded RNA initiates a post-transcriptional genic silencing process, through degradation of homologous mRNAs, causing a decrease in the synthesis of the corresponding protein (MEISTER, G.; TUSCHL, T., Mechanisms of gene silencing by double-stranded RNA. Nature, v. 431, n. 7006. p. 343-349. 2004), making the survival difficult or even leads the parasite to death.
Ever since its initial description, this technique has become a valuable tool for the functional genomics of insects, in particular in the study of the genic function on Drosophila melanogaster (MESQUITTA, L.; PATERSON, B. M., Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA, v. 96, n. 4. p. 1451-1456. 1999; KENNERDELL, J. R.; CARTHEW, R. W., Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol, v. 18, n. 8. p. 896-898. 2000). Recent Works showed the feasibility of plants that used to produce dsRNA in resistance against pest insects. These transgenic plants produced specific dsRNA against essential genes in the digestive tract of the insects, causing mortality in 24 hours after contact with the RNAi (BAUM, J. A., et al., Control of coleopteran insect pests through RNA interference. Nat Biotechnol, v. 25, n. 11. p. 1322-1326. 2007; MAO, Y. B., et al., Silencing a cotton bollworm P450 monooxygenase gene by plant mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol, v. 25, n. 11. p. 1307-1313. 2007).
The application of RNAi in planta has a great potential as an approach to handling insects. The specificity of the RNAi for insecticidal purposes is an important consideration for the use of this technology in practical applications, since the effects on non-target insects may be minimized. Among other advantages, this technique enables the use of only sequence fragments, since the translation of a protein is not necessary, which minimizes the worries about biosafety and allergenicity, and represents a probably more effective form of control than the present-day ones.
Chitin, a linear polysaccharide formed by residues of N-acetyl-D-glycosamine United by β (1-4) links, is widespread among insects, which use this versatile biopolymer in various anatomic structures. Two main extracellular structures where the deposition of chitin takes place are the cuticle that covers the epidermis and the peritrophic membrane that covers the middle intestine (MUTHUKRISHNAN, S., et al., Chitin Metabolism in Insects. In Insect Molecular Biology and Biochemistry, 1 ed.; Gilbert, L. I., Ed. Elsevier: London, pp. 193-225. 2012).
The peritrophic membrane is a functional structure that covers the middle intestine of the insects. The main functions attributed to this membrane are that of mechanical protection against injury to cells of the middle intestine (WIGGLESWORTH, V., The principles of insect physiology. 7 ed.; Chapman and Hall: London, Vol. p 827. 1972), a physical barrier against microorganisms (PETERS, W., Peritrophic membranes. Springer-Verlag New York, Vol. 1992), a barrier selective for digestive enzymes and digestion products (DAY, M. F.; WATERHOUSE, D. F., Functions of the alimentary system. John Wiley: New York, Vol. p 299-310. 1953) and actuation as a mechanism of recycling digestive enzymes, a phenomenon known as ectoendoperitrophic circulation (TERRA, W. R., Physiology and Biochemistry of Insect Digestion—an Evolutionary Perspective. Brazilian Journal of Medical and Biological Research, v. 21, n. 4. p. 675-734. 1988; TERRA, W. R.; FERREIRA, C., Insect Digestive Enzymes—Properties, Compartmentalization and Function. Comparative Biochemistry and Physiology Biochemistry & Molecular Biology, v. 109, n. 1. p. 1-62. 1994; TERRA, W. R., The origin and functions of the insect peritrophic membrane and peritrophic gel. Archives of Insect Biochemistry and Physiology, v. 47, n. 2. p. 47-61, 2001).
The insect cuticle or exoskeleton is a multifunctional structure that serves as a physical support, and also gives its shape, enables displacement, imparts impermeability to the body, and a number of localized mechanic specializations, such as high degree of adhesion, resistance to wear and diffusion control. In this structure, its mechanical properties are attributed to its main constituent, chitin (VINCENT, J. F., et al., Design and mechanical properties of insect cuticle. Arthropod Struct Dev, v. 33, n. 3. p. 187-199. 2004).
The synthesis and deposition of chitin on the cuticle and on the peritrophic membrane comprise a sequential series of complex biochemical, biophysical, intracellular and extracellular transformations, some of which still little understood (MOUSSIAN, B., et al., Assembly of the Drosophila larval exoskeleton requires controlled secretion and shaping of the apical plasma membrane. Matrix Biol, v. 26, n. 5. p. 337-347. 2007). Since chitin is absent in plants and vertebrates, its biosynthetic pathway is one of the main targets for the development of insecticides ever since 1970 (VERLOOP, A., et al., Benzoylphenyl Ureas—A New Group of Larvicides Interfering with Chitin Deposition. In Pesticide Chemistry in the 20th Century, AMERICAN CHEMICAL SOCIETY: Vol. 37, pp. 237-270. 1977). Among the enzymes involved in the synthesis of chitin on insects, a special approach has been given to the last step of the pathway, which is measured by the enzyme chitin synthase (EC 2.4.1.16), which catalyzes the polymerization of chitin from activated monomers of UDP-N-acetilglicosamina (MERZENDORFER, H., The cellular basis of chitin synthesis in fungi and insects: common principles and differences. Eur J Cell Biol, v. 90, n. 9. p. 759-769. 2011).
The interruption or the decrease in the synthesis of enzymes that participate in the biosynthesis of constituents of the cuticle and of the peritrophic membrane of the insect by means of the RNAi technology proves to be a specific form of control of pest insects.
Chitin synthase A (or type 1) on insects is the main enzyme involved in the biosynthesis of chitin of the cuticle and of the trachea. However, chitin synthase B (or type 2) on insects is the main enzyme involved in the biosynthesis of chitin of the peritrophic membrane, both chitins synthases of insects have been studied as ideal targets for the development of strategies of control of pest insects.
In the present invention, one shows the insecticidal effect of dsRNA molecules on the “bicudo-do-algodoeiro” or boll weevil (Anthronomus grandis), the main pest of cotton-plantation in Brazil. The insecticidal effect is attributed to the inhibition of the transcript levels of the enzymes chitin synthase 1 and 2, which caused damages to the development of eggs and adults of A. grandis when subjected to the dsRNA treatment against these enzymes.